Project Summary/Abstract: Role of O-GlcNAc Transferase-Mediated Innate Immune Response in Colonic Inflammation and Tumorigenesis Ulcerative colitis is a common form of inflammatory bowel disease that affects about 1 person per 600 in the U.S. Patients suffering from colitis demonstrate a significantly increased risk of colorectal cancer (CRC). A common feature of colitis and CRC is enhanced activation of immune signaling and inflammatory cytokine production. Studies of immune system metabolism (immunometabolism) in recent years have identified a tight link between metabolic reprogramming and inflammation. It has been shown that activation of immune cells is accompanied by robust metabolic changes towards increased glucose utilization. However, the role of individual glucose metabolism pathways in colonic inflammation and tumorigenesis remains unknown. The hexosamine biosynthesis pathway is a unique glucose metabolism pathway that leads to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). O-linked GlcNAc (O-GlcNAc) transferase (OGT) is a key enzyme that utilizes UDP-GlcNAc to modify its target proteins, a process called O-GlcNAcylation. In this Proposal, we aim to study the role of O-GlcNAc signaling in innate immune activation of colonic inflammation and tumorigenesis. Our preliminary studies revealed increased OGT and decreased cullin 3 (Cul3) E3 ubiquitin ligase expression in inflamed colon tissue during chemically-induced colitis. Deletion of OGT or Cul3 in myeloid cells attenuated or exacerbated disease severity during colitis, respectively. The underlying mechanism may involve O-GlcNAcylation-mediated inhibition of signal transducer and activator of transcription 3 (STAT3), a central transcription factor in the regulation of intestinal inflammation and tumorigenesis. Through microarray and biochemical analyses, our recent study (JEM, In Press) identified Cul3 as a negative regulator of OGT expression and STAT3 O-GlcNAcylation by inhibiting nuclear factor E2-related factor-2 (Nrf2). Our studies further revealed lysine-63 (K63)-linked ubiquitination of OGT mediated by TNF receptor associated factor 6 (TRAF6), a crucial E3 ubiquitin ligase regulating immune signaling. Based on the known effects of STAT3 in colitis and CRC, and the well-established cross-regulation between O-GlcNAcylation and phosphorylation, we propose that altered signaling through a Cul3-OGT axis leading to aberrant STAT3 O- GlcNAcylation plays an essential role in innate immune activation during colitis and associated colon cancer. We hypothesize that 1) increased OGT expression and STAT3 O-GlcNAcylation in myeloid cells exacerbates colonic inflammation and inflammation-driven colon cancer; 2) myeloid-derived Cul3 negatively regulates OGT expression and promotes activation of a STAT3-IL-10 axis; and 3) TRAF6-mediated OGT K63 ubiquitination is important for STAT3 O-GlcNAcylation. We will employ both a chemically-induced model and the IL-10 deficiency model in mice to investigate acute colitis and inflammation-driven colon cancer. It is our expectation that this work will bring about a better understanding of metabolic regulation of intestinal inflammation and tumorigenesis and contribute to the generation of new strategies for the treatment of these diseases.